(i) Field of the Invention
The invention relates to a regulatory nucleotide sequence of the initiation of transcription and its use in the production of polypeptides by the recombinant approach. The technical problem which presented itself when the present invention was being developed was to identify a strong and, if possible, thermoinducible promoter, which could be used in a large number of micro-organisms and, in particular, in the Actinomycetes.
(ii) Description of Related Art
The Actinomycetes constitute a bacterial order of great economic and medical importance, mention needs only to be made of the fact that the Actinomycetes include, in particular, the Streptomyces and Mycobacterium genera.
The Streptomyces are used for the production of about 70% of the antibiotics sold today; furthermore, even though there is no longer occasion to describe the ravages caused by Mycobacterium tuberculosis and Mycobacterium leprae, great interest is attached to the expression of the heterologous antigen in a strain of M. bovis BCG in order to produce a living polyvalent strain to be used as a vaccine.
The genetics of these bacteria has been little studied at the molecular level, but the regulation of genetic expression seems to be different from that which has been described for bacteria which have been studied in more detail such as Escherichia coli or Bacillus subtilis.
In particular, the Actinomycetes, the DNA of which is rich in G+C, recognize the "promoter" sequences in Escherichia coli and Bacillus subtilis, poorly or not at all. Baird et al. (J. Gen. Microbiol.1989, 135, 931-939) studied the genes coding for heat shock proteins in Mycobacterium and postulated that two sequences, TTGAG and TCTCATGT, located upstream from the sequence coding for the heat shock protein of 10 kDA of Mycobacterium tuberculosis, constitute the -35 and -10 regions of the promoter.
In fact, the two sequences exhibit a high degree of homolgy with the consensus sequence of the promoter in E. coli. Furthermore, sequences located upstream from the genes coding for other heat shock proteins in various species of Mycobacterium (for example, the protein of 65 kDa of Mycobacterium leprae or that of 64 kDA in M. bovis) also contain a couple of sequences of the same type, i.e.: TFGCCG and TTTCAT, or TTGCCG and CTTCAT, and which thus show a high degree of homology with the E. coli promoter and with the "-35 and -10" sequences of the 10 kDa species of Mycobacterium tuberculosis.
According to that article and the one by Thole et al. (Infection and Immunity, 55, 1987, 1466-1475), the promoters responsible for the transcription of the genes of the heat shock proteins in Mycobacterium contain, as -35 and -10 sequences, this type of coupled sequences. These sequences will be designated subsequently as "-10 and -35 sequences of the E. coli type". This being so, the authors had not confirmed this hypothesis by mapping and the identity of the promoters consequently remains uncertain.
In a surprising manner, the present inventors have noticed that the -10 and -35 sequences of the E. coli type are also present in Streptomyces, but do not play a role in the initiation of the transcription of heat shock proteins in the bacteria of this genus. It should be noted that, in the articles mentioned above, Baird and Thole had also observed the presence, in sequences upstream from the genes coding for the heat shock proteins in Mycobacterium, a palindromic motif containing the sequence (SEQ ID NO:1) GCACTC 9N GAGTGC. However, the precise role of this motif had not been identified. Thole postulates that this motif is implicated either in the termination of transcription of an operon located upstream from the gene coding for the heat shock protein or in the regulation of the translation of a polycistronic messenger.
With the aim of cloning a strong (and, if possible, thermoinducible) promoter, which can be used in a large number of Actinomycetes, the inventors have studied the response to heat shock in Streptomyces and have cloned a strongly expressed protein in order to characterize its promoter. In fact, the response phenomenon to heat shock resulting in the de novo of proteins is a universal phenomenon; it may thus be anticipated that its regulation will be similar in various Actinomycetes and in particular that the promoters will have consensus sequences which will enable them to be used in a large number of strains.